Biofuels are under intensive investigation due to the increasing concerns about energy security, sustainability, and global climate change (Lynd et al., 1991). Bioconversion of plant-derived lignocellulosic materials into biofuels has been regarded as an attractive alternative to chemical production of fossil fuels (Lynd et al. 2008; Hahn-Hagerdal et al. 2006). Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin.
The engineering of microorganisms to perform the conversion of lignocellulosic biomass to ethanol efficiently remains a major goal of the biofuels field. Much research has been focused on genetically manipulating microorganisms that naturally ferment simple sugars to alcohol to express cellulases and other enzymes that would allow them to degrade lignocellulosic biomass polymers and generate ethanol within one cell. However, an area that has been less well studied is that of sugar transporters. An understanding of the regulation of sugar transport and the genetic engineering of microorganisms to have improved sugar-uptake ability will greatly improve efficiency (Stephanopoulos 2007). Furthermore, other types of proteins involved in the regulation of cellulase expression and activity remain to be fully explored.
Saccharomyces cerevisiae, also known as baker's yeast, has been used for bioconversion of hexose sugars into ethanol for thousands of years. It is also the most widely used microorganism for large scale industrial fermentation of D-glucose into ethanol. S. cerevisiae is a very suitable candidate for bioconversion of lignocellulosic biomass into biofuels (van Maris et al., 2006). It has a well-studied genetic and physiological background, ample genetic tools, and high tolerance to high ethanol concentration and inhibitors presented in lignocellulosic hydrolysates (Jeffries 2006). The low fermentation pH of S. cerevisiae can also prevent bacterial contamination during fermentation.
Unfortunately, wild type S. cerevisiae cannot utilize pentose sugars (Hector et al., 2008). To overcome this limitation, pentose utilization pathways from pentose-assimilating organisms have been introduced into S. cerevisiae, allowing fermentation of D-xylose and L-arabinose (Hahn-Hagerdal et al., 2007; Brat et al., 2009; Wisselink et al., 2007, 2009; Wiedemann and Boles 2008; Karhumma et al., 2006). However, efficient conversion of pentose sugars into biofuels is limited by multiple issues including cellular redox imbalance, low influx of pentose phosphate pathway, and lack of efficient pentose transport into the cell (Hector et al., 2008).
In addition, both natural and engineered microorganisms show reduced ethanol tolerance during xylose fermentation as compared to glucose fermentation (Jeffries and Jin 2000). Combined with the lower fermentation rate, the reduced ethanol tolerance during xylose fermentation poses a significant problem in fermentation of sugar mixtures containing the high concentrations of glucose (˜70-100 g/L) and xylose (˜40-60 g/L) present in cellulosic hydrolysates. Since microorganisms utilize glucose preferentially, at the time of glucose depletion (when cells begin to use xylose), the ethanol concentration is already high enough (˜35-45 g/L) to further reduce the xylose fermentation rate. As a result, sequential utilization of xylose after glucose depletion because of “glucose repression” is a significant challenge to be overcome in order to successfully utilize mixed sugars in cellulosic hydrolysates.
Thus, a need exists for the identification of additional genes that are critical for the degradation of lignocellulose and for their use in the engineering of microorganisms for improved growth on lignocellulose and uptake of compounds resulting from lignocellulose degradation. A further need exists for improved methods of efficient conversion of pentose sugars into biofuels and of mixed sugar fermentation for the production of biofuels.